Takeshi Kashimoto

Effects of ginseng saponins on responses induced by various receptor stimuli

We investigated the effects of four ginseng saponins, ginsenoside-Rb1, -Rg2, -Rg3 and -Ro, on the responses induced by receptor stimulation of various stimuli. Ginsenoside-Rg2 (1-100 microM) reduced the secretions of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine and gamma-aminobutyric acid but not by angiotensin II, bradykinin, histamine and neurotensin. In guinea-pig, the ginsenoside also diminished the nicotine-induced secretion of catecholamines from the adrenal chromaffin cells, but it did not affect the muscarine- and the histamine-induced ileum contractions. On the other hand, ginsenoside-Rg3 (1-100 microM) reduced not only the acetylcholine-, the gamma-aminobutyric acid- and the neurotensin-induced secretions but also, at a higher concentration (100 microM), the angiotensin II-, the bradykinin- and the histamine-induced secretions from the bovine chromaffin cells. Furthermore, the saponin (3-100 microM) significantly inhibited the muscarine- and the histamine-induced ileum contractions of the guinea-pig. Ginsenoside-Rb1 and -Ro had no marked effect on their responses. These results strongly suggest that ginsenoside-Rg2 is a potent selective blocker of nicotinic acetylcholine and gamma-aminobutyric acid receptors (ionotropic receptors) and ginsenoside-Rg3 is not only a blocker of ionotropic receptors but also an antagonist of muscarinic or histamine receptors.

Properties of ginseng saponin inhibition of catecholamine secretion in bovine adrenal chromaffin cells

To investigate the relationship between the inhibitory effects of ginseng saponins (ginsenosides) on acetylcholine-evoked secretion of catecholamines and the structures of ginsenosides, we examined the effects of ginsenoside-Rg3 and -Rh2, which are panaxadiol saponins, 20(R)- and 20(S)-ginsenoside-Rg2, which are epimers involving the hydroxyl group at C-20 of sapogenin, and other plant saponins on the acetylcholine-evoked secretion of catecholamines from cultured bovine adrenal chromaffin cells. The ginsenoside-Rg3 (1-100 microM) and -Rh2 (10-100 microM) greatly reduced the acetylcholine-evoked secretion in a concentration-dependent manner comparable to that of ginsenoside-Rg2, a panaxatriol saponin, which was the most potent inhibitor in our previous study. 20(R)- and 20(S)-ginsenoside-Rg2 (1-100 microM) similarly reduced the acetylcholine-evoked secretion. In contrast, saikosaponin-a, glycyrrhizin and the cardiac glycosides (100 nM-100 microM), digitoxin and digoxin, had no significant inhibitory effect on catecholamine secretion. Saikosaponin-c (10-100 microM), however, had an inhibitory effect, which was less than that of ginsenoside-Rg2 and -Rg3. These results strongly suggest that the inhibitory effects of ginsenosides on the acetylcholine-evoked secretion of catecholamines from bovine adrenal chromaffin cells are a unique property of ginseng. Further, the relationship between the inhibitory effects and the structures of ginsenosides is discussed.

Effects of extract and ingredients isolated from Magnolia obovata Thunberg on catecholamine secretion from bovine adrenal chromaffin cells.

The crude extract of magnolia bark, an herbal drug, inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh) in a concentration-dependent manner (200-900 microg/mL). The extract also diminished the secretion induced by high K(+), which is a stimulus directly depolarizing the plasma membranes, but its inhibition was weaker than that of ACh-evoked secretion. beta-Eudesmol, honokiol, magnolol, and bornyl acetate, but not alpha- and beta-pinenes, all of which are ingredients of magnolia bark, greatly reduced ACh-evoked secretion. beta-Eudesmol and magnolol also inhibited high K(+)-induced secretion to an extent similar to that of ACh-evoked secretion. However, honokiol and bornyl acetate inhibited the secretion induced by high K(+) much less than the secretion evoked by ACh. ACh-induced Na(+) influx and ACh- or high K(+)-induced Ca(2+) influx into the cells were diminished by beta-eudesmol or honokiol. These results indicate that magnolia bark contains some effective components inhibiting the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by ACh due to the antagonism of Na(+) and Ca(2+) influxes into the cells. However, inhibition by the extract of magnolia bark seems to be attributable to honokiol and bornyl acetate. Furthermore, the results indicate that the inhibitory effect of magnolia bark may be associated with its pharmacological effect on activities of the nervous system.

Down-Regulation of the Noradrenaline Transporter by Interferon-α in Cultured Bovine Adrenal Medullary Cells

Abstract: The aim of this study was to investigate the effect of long‐term treatment with interferon (IFN)‐α on the noradrenaline transporter of bovine adrenal medullary cells. Treatment of cultured adrenal medullary cells with IFN‐α caused a decrease in uptake of [3H]noradrenaline by the cells in time (4–48 h)‐ and concentration (300–1,000 U/ml)‐dependent manners. IFN‐β also inhibited [3H]noradrenaline uptake to a lesser extent than did IFN‐α, whereas IFN‐γ had little effect. An anti‐IFN‐α antibody reduced the effect of IFN‐α on [3H]noradrenaline uptake. Saturation analysis of [3H]noradrenaline uptake showed that the inhibitory effect of IFN‐α was due to a reduction in the maximal uptake velocity (Vmax) values without altering apparent Michaelis constant (Km) values. Incubation of cells with IFN‐α caused a translocation of protein kinase C from the soluble to the particulate fraction in the cells. The effect of IFN‐α on [3H]noradrenaline uptake was diminished in protein kinase C‐down‐regulated cells. Incubation of cells with IFN‐α for 48 h significantly reduced the specific binding of [3H]desipramine to crude plasma membranes isolated from cells. Scatchard analysis of [3H]desipramine binding revealed that IFN‐α decreased the maximal binding (Bmax) values without any change in the dissociation constant (KD) values. These findings suggest that IFN‐α suppresses the function of noradrenaline transporter by reducing the density of the transporter in cell membranes through, at least in part, a protein kinase C pathway.

Characterization of ginseng saponin ginsenoside-Rg3 inhibition of catecholamine secretion in bovine adrenal chromaffin cells

Since ginsenoside-Rg(3), one of the panaxadiol saponins isolated from the ginseng root, significantly inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), the properties of ginsenoside-Rg(3) inhibition were investigated. Although ginsenoside-Rg(3) inhibited the secretion evoked by ACh in a concentration-dependent manner, it affected the secretion stimulated by high K(+) or veratridine, an activator of the voltage-sensitive Ca(2+) or Na(+) channels, only slightly. The ACh-induced Na(+) and Ca(2+) influxes into the cells were also reduced by ginsenoside-Rg(3). The inhibitory effect of this saponin on the secretion of catecholamines was not altered by increasing the external concentration of ACh or Ca(2+). The ACh-evoked secretion of catecholamines was completely restored in cells that were preincubated with 10 microM ginsenoside-Rg(3) and then incubated without the saponin, whereas secretion was not completely restored in cells that were preincubated with 30 microM of this compound. Above 30 microM ginsenoside-Rg(3) increased the fluorescence anisotropy of diphenylhexatriene in the cells. Furthermore, the inhibitory effect of ginsenoside-Rg(3) at 30 microM on the ACh-evoked secretion of catecholamines was dependent upon the preincubation time, but this was not the case at 10 microM. These results strongly suggest that ginsenoside-Rg(3) blocks the nicotinic ACh receptor-operated cation channels, inhibits Na(+) influx through the channels, and consequently reduces both Ca(2+) influx and catecholamine secretion in bovine adrenal chromaffin cells. In addition to this action, the ginsenoside at higher concentrations modulates the fluidity of the plasma membrane, which probably contributes to the observed reduction in the secretion of catecholamines.

Mechanism of the Effect of Droperidol to Induce Catecholamine Efflux from the Adrenal Medulla

The study was undertaken to determine whether droperidol had an effect to induce catecholamine efflux from the adrenal medulla as a mechanism for the possible pressor effect of droperidol in patients with pheochromocytoma and, if so, to ascertain the site of action of this compound. The efflux of catecholamines from perfused dog adrenals was increased from control level, 0.15 μg/min, to 0.66 μg/min by the administration of droperidol 6.6 μm. This effect of droperidol was not dependent on extracellular Ca++, in contrast to acetylcholine. The concomitant secretion of catecholamines and dopamine-β-hydroxylase was observed in response to acetylcholine and caffeine. However, droperidol-, histamine-, and reserpine-induced catecholamine efflux was not accompanied by dopamine-ß-hydroxylase release. In additional studies, chromaffin granules were isolated with a Millipore· filter technique from the bovine adrenal medulla and were incubated for 10 min in an isotonic medium to examine the direct effects of droperidol. Droperidol did not enhance the efflux of catecholamines from the granules in contrast to histamine. The upake of 14C-norepinephrine into the granules was inhibited by droperidol in a manner comparable to reserpine. The results suggest that droperidol induces catecholamine efflux from adrenal medullary cells and the efflux probably is caused by a nonexocytotic mechanism. A contributing mechanism was an inhibition of catecholamine uptake into chromaffin granules, resulting in an increased diffusion of catecholamines out of the cell.

p-chloromercuribenzoate causes Ca2+-dependent exocytotic catecholamine secretion from cultured bovine adrenal medullary cells

Abstract: Incubation of cultured bovine adrenal medullary cells with p‐chloromercuribenzoate (50–500 μM), a sulfhy‐dryl‐reacting agent, caused an increase in the secretion of catecholamines. p‐Chloromercuriphenyl sulfonate, a p‐chlo‐romercuribenzoate analogue that poorly penetrates the cell membrane, caused a similar increase in catecholamine secretion. In both cases, catecholamine secretion was dependent on extracellular Ca2+. Furthermore, p‐chloromercuribenzoate caused both 45Ca2+ influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and 45Ca2+ influx behaved similarly in relation to p‐chloromercuribenzoate concentration. The time courses of the increased secretion, 45Ca2+ influx, and intracellular free Ca2+ concentration by p‐chloromercuri‐benzoate were also quite similar. The stimulation of cate‐holamine secretion by p‐chloromercuribenzoate was reversed by washing the cells with dithiothreitol‐containing medium, but not by dithiothreitol‐free medium. When the cells were treated with p‐chloromercuribenzoate, dopamineβ‐hydroxylase, an enzyme present in the chromaffin granules along with catecholamines, was also released. However, p‐chloromercuribenzoate did not cause release of phenyletha‐nolamine‐JV‐methyltransferase, an enzyme present in the cytoplasm. These results indicate that catecholamine secretion due to p‐chloromercuribenzoate occurs by Ca2+‐dependent exocytosis.

INTERFERON-ALPHA REDUCES CATECHOLAMINE SECRETION FROM BOVINE ADRENAL CHROMAFFIN CELLS STIMULATED BY ACETYLCHOLINE

A long-term pretreatment (72h) of bovine adrenal chromaffin cells with recombinant human interferon (IFN)-α-2b (1500 units/ml) produced a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine (ACh) (25μmol/l) but not that with human fibloblast IFN-β (3000 units/ml) or recombinant human IFN-γ (3000 units/ml). IFN-α-2b inhibited the ACh-induced secretion in a concentration- (30–1500 units/ml) and time-dependent manner (18–72h). The content of catecholamines in the cells treated with IFN-α-2b for 72h did not change. The inhibitory effect of IFN-α-2b on the secretion was abolished when the cells were simultaneously treated with anti-IFN-α antibody, and it was overcome by the increase in the external ACh concentration. IFN-α-2b also inhibited ACh-induced Ca2+ influx into the cells in a concentration-dependent manner similar to that of the IFN-α-2b inhibiting ACh-induced secretion. On the other hand, IFN-α-2b failed to reduce the secretion from the cells induced by high K+. These results strongly suggest that IFN-α-2b reduces the ACh-induced secretion of catecholamines from bovine adrenal chromaffin cells due to modulating the gene expression of the nicotinic ACh receptor-operated cation channels rather than due to directly affecting the channels. The results further indicate that the IFN-α-2b inhibition may be associated with the psychiatric side effects of IFN-α (depression, neurasthenica and somnolence, etc.), and that immune systems may regulate the function of (autonomic) nervous systems or adrenal medulla via IFN-α in vivo.

Inflammatory cytokines decrease the expression of nicotinic acetylcholine receptor during the cell maturation

It is known that the nervous system significantly attenuates systemic inflammatory responses through the parasympathetic nervous system. Furthermore, it has been reported that the alpha7 subunit of a nicotinic acetylcholine receptor is required for a cholinergic inhibition against cytokine synthesis in a macrophage. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses and the maintenance of immunity, in this study, we investigated the expression level of nicotinic receptors of a p53-deficient APC cell line (JawsII) derived from a mouse bone marrow. We showed that stimulation of the JawsII cells with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α) led increase of CD80 and CD86 expression while diminishment of the surface nicotinic receptor. On the other hand, stimulation of nicotinic receptor had no effect on these phenomena. Furthermore, we examined the ability of the cells to release cytokine when stimulated with both nicotine and LPS and showed that the stimulation with LPS augmented the secretion of IL-1a, IL-1b, IL-6, and TNF-α. These results suggested that nicotinic stimulation had no effect on the diminishment of alpha7 nicotinic acetylcholine receptor on JawsII cells by LPS stimulation.

Inhibition by pregnenolone sulfate of nicotinic acetylcholine response in adrenal chromaffin cells

To evaluate whether pregnenolone sulfate, an abundant neurosteroid in the brain, modulates nicotinic receptor-mediated responses, the effect of pregnenolone sulfate on acetylcholine-induced catecholamine secretion was investigated in cultured bovine adrenal chromaffin cells. Pregnenolone sulfate inhibited acetylcholine-induced catecholamine secretion (IC(50): 27 microM). In addition, pregnenolone sulfate inhibited acetylcholine-induced Na(+) (IC(50): 12 microM) and Ca(2+) (IC(50): 20 microM) influxes. However, pregnenolone sulfate did not inhibit either catecholamine secretion or Ca(2+) influx stimulated by high K(+). Binding of [3H]nicotine to nicotinic receptors was not altered by pregnenolone sulfate. The inhibitory effect on the acetylcholine-induced secretion was insurmountable by increasing acetylcholine concentrations, but was enhanced by decreasing external Na(+) concentrations. These results suggest strongly that pregnenolone sulfate noncompetitively inhibits nicotinic receptor-operated ion channels, thereby suppressing Na(+) influx through the channels and, consequently, attenuates both Ca(2+) influx and catecholamine secretion. Our results further indicate that pregnenolone sulfate may modulate nicotinic receptor-mediated responses in the brain.